title: Identifying key amino acid residues that affect α-conotoxin AuIB inhibition of α3β4 nicotinic acetylcholine receptors.
abstract: α-Conotoxin AuIB is a selective α3β4 nicotinic acetylcholine receptor (nAChR) subtype inhibitor. Its analgesic properties are believed to result from it activating GABAB receptors and subsequently inhibiting CaV2.2 voltage-gated calcium channels. The structural determinants that mediate diverging AuIB activity at these targets are unknown. We performed alanine scanning mutagenesis of AuIB and α3β4 nAChR, homology modeling, and molecular dynamics simulations to identify the structural determinants of the AuIB·α3β4 nAChR interaction. Two alanine-substituted AuIB analogues, [P6A]AuIB and [F9A]AuIB, did not inhibit the α3β4 nAChR. NMR and CD spectroscopy studies demonstrated that [F9A]AuIB retains its native globular structure, so its activity loss is probably due to loss of specific toxin-receptor residue pairwise contacts. Compared with AuIB, the concentration-response curve for inhibition of α3β4 by [F9A]AuIB shifted rightward more than 10-fold, and its subtype selectivity profile changed. Homology modeling and molecular dynamics simulations suggest that Phe-9 of AuIB interacts with a two-residue binding pocket on the β4 nAChR subunit. This hypothesis was confirmed by site-directed mutagenesis of the β4-Trp-59 and β4-Lys-61 residues of loop D, which form a putative binding pocket. AuIB analogues with Phe-9 substitutions corroborated the finding of a binding pocket on the β4 subunit and gave further insight into how AuIB Phe-9 interacts with the β4 subunit. In summary, we identified critical residues that mediate interactions between AuIB and its cognate nAChR subtype. These findings might help improve the design of analgesic conopeptides that selectively "avoid" nAChR receptors while targeting receptors involved with nociception.
authors.affiliation: From the Health Innovations Research Institute, RMIT University, Melbourne, Victoria 3083, Australia and.
authors.forename: Anton A
authors.initials: AA
authors.lastname: Grishin
authors.forename: Hartmut
authors.initials: H
authors.lastname: Cuny
authors.forename: Andrew
authors.initials: A
authors.lastname: Hung
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Clark
authors.forename: Andreas
authors.initials: A
authors.lastname: Brust
authors.forename: Kalyana
authors.initials: K
authors.lastname: Akondi
authors.forename: Paul F
authors.initials: PF
authors.lastname: Alewood
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
authors.forename: David J
authors.initials: DJ
authors.lastname: Adams
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D020864:Calcium Channels, N-Type / Q000737:chemistry / Q000378:metabolism; D020916:Conotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D059225:Nociception; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D000596:Amino Acids; C500545:CACNA1B protein, human; D020864:Calcium Channels, N-Type; D020916:Conotoxins; D011978:Receptors, Nicotinic; C115558:conotoxin AuIB; C403530:nicotinic receptor alpha3beta4; D000409:Alanine
keywords: Cyonotoxin; Electrophysiology; Molecular Docking; Molecular Modeling; NMR; Nicotinic Acetylcholine Receptors; Peptide Chemical Synthesis; Site-directed Mutagenesis; Xenopus Oocyte